Background— Current methods of MSC cryopreservation result in variable post thaw recovery and phenotypic changes due to freezing. The objective of this investigation is to determine the influence of ex-vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. Methods— Human bone marrow aspirate was purchased from Lonza (Walkersville, MD). MSCs were isolated, and cells were assessed for total count, viability, apoptosis, and senescence over 6 passages (8–10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were re-plated for continued culture, and the other half were frozen at 1°C/min in a controlled rate freezer. Frozen samples were stored in liquid nitrogen, thawed, and reassessed for total cell count, viability, and senescence immediately and 48 hours post thaw. Results— Viability did not differ significantly between samples pre freeze or post thaw. Senescence increased over time in pre freeze culture, and was significantly higher in one sample that experienced growth arrest both pre freeze and post thaw. Freezing resulted in similar initial post thaw recovery in all samples, but 48 hour post thaw growth arrest was observed in the sample with high senescence only. Conclusion— High freeze senescence appears to correlate with poor post thaw function in MSC samples, but additional studies are necessary to obtain a sample size large enough to quantify results. MSCs had reduced immunomodulatory and blood regulatory properties immediately post thaw. 12 These temporal and freezing induced changes in cell behavior can lead to confounding outcomes for clinical studies using cryopreserved MSCs. One investigator hypothesizes that poor post thaw MSC function may have been responsible for the failure of a recent clinical trial. 13 The objective of this investigation is to determine the influence of ex vivo cell expansion on phenotype of MSCs at harvest and the response of resulting phenotypes to freezing and thawing. This information will help clarify the influence of culture conditions on the biological characteristics of MSC products and potential shifts in composition or behavior resulting from the freezing process.

Pre-clinical data showed that C3a priming of CD34+ HPC leads to activation of CXCR4, improved migration towards an SDF1 gradient, and faster hematopoietic recovery and better human engraftment in mice. On the basis of these data, we hypothesized that preincubation with C3a would be safe and enhance engraftment. To test this hypothesis, we incubated the smaller of the two UCB units with C3a 1 mcg/mL (Calbiochem, Gibbstown, NJ) for 30 minutes, and infused without washing. Ten patients, median age 62 (r: 30-68), 8 males, with AML (n = 6), MDS (n = 3) and Hodgkin's (n = 1) were enrolled. Units were selected using the University of Minnesota published criteria and all met lot release (endotoxin <5 EU/kg, negative gram stain, viability >45%). Conditioning consisted of cyclophosphamide 50 mg/kg/x1day, fludarabine 40 mg/m2/x5d, total body irradiation 200cGy/x1d with antithymocyte globulin added in three since they had not had recent chemotherapy. Immunoprophylaxis consisted of cyclosporine A/mycophenolate mofetil (MMF). The unmanipulated unit was infused first with the C3a primed unit infused 30 min later. Patients were monitored for 24 h for evidence of infusional toxicities and activation of pathways downstream of C3a by assessment of coagulation factors, IL-6, TNF-alpha, histamine, tryptase and C-reactive protein before and 15, 30 and 60 min post-infusion. Grade 3 hypertension was the only notable infusional toxicity in 5; no patient had demonstrable activation of the complement pathway. Nine patients had neutrophil recovery at a median of 9 days (range 6-26). On day 21, hematopoiesis was primarily derived from the C3a primed unit in 6 of 9 (67%) evaluable patients and present but non-predominating in another. While not expected to enhance the speed of neutrophil recovery after a non myeloablative therapy where initial host recovery in expected, these data suggest a potential repopulating advantage especially since the C3a primed unit is smaller and infused last. Based on the safety profile and potential impact on engraftment, a randomized phase II clinical trial in the myeloablative setting where neutrophil recovery rates are suboptimal (median 23 days) is being developed.

TransfusionVolume 49, Issue 5 p. 1018-1019 Transfusion-associated graft-versus-host disease: a perspective from a cell therapy laboratory D. Hummon, D. Hummon University of Notre DameNotre Dame, INSearch for more papers by this authorN.D. Zantek MD, PhD, N.D. Zantek MD, PhD Division of Transfusion MedicineSearch for more papers by this authorD. Sumstad MT, CLS, D. Sumstad MT, CLS Division of Transfusion MedicineSearch for more papers by this authorJ.S. Miller MD, J.S. Miller MD Blood and Marrow Transplant ProgramSearch for more papers by this authorD.H. McKenna MD, D.H. McKenna MD e-mail: mcken020@umn.eduDivision of Transfusion MedicineUniversity of Minnesota Medical SchoolMinneapolis, MNSearch for more papers by this author D. Hummon, D. Hummon University of Notre DameNotre Dame, INSearch for more papers by this authorN.D. Zantek MD, PhD, N.D. Zantek MD, PhD Division of Transfusion MedicineSearch for more papers by this authorD. Sumstad MT, CLS, D. Sumstad MT, CLS Division of Transfusion MedicineSearch for more papers by this authorJ.S. Miller MD, J.S. Miller MD Blood and Marrow Transplant ProgramSearch for more papers by this authorD.H. McKenna MD, D.H. McKenna MD e-mail: mcken020@umn.eduDivision of Transfusion MedicineUniversity of Minnesota Medical SchoolMinneapolis, MNSearch for more papers by this author First published: 21 April 2009 https://doi.org/10.1111/j.1537-2995.2009.02121.xCitations: 6Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume49, Issue5May 2009Pages 1018-1019 RelatedInformation

BACKGROUND:Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. METHODS:Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS:Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION:The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

BACKGROUND:Natural killer (NK) cells, a subset of lymphocytes and part of the innate immune system, play a crucial role in defense against cancer and viral infection. Herein is a report on the experience of clinical-scale, good manufacturing practices (GMPs) production of NK cells to treat advanced cancer.STUDY DESIGN AND METHODS:Two types of NK cell enrichments were performed on nonmobilized peripheral blood mononuclear cell apheresis collections with a cell selection system (CliniMACS, Miltenyi): CD3 cell depletion to enrich for NK cells and CD3 cell depletion followed by CD56 cell selection to obtain a more pure NK cell product. After overnight incubation with interleukin-2 (IL-2), cells were washed, resuspended in 5 percent human serum albumin, and then released for infusion.RESULTS:A total of 70 NK cell therapy products have been manufactured for patient infusion since 2000. For the CD3 cell-depleted NK cell products, the mean purity, recovery, and viability were 38, 79, and 86 percent, respectively. For the CD3 cell-depleted/CD56 cell-enriched NK cell products, the mean purity, recovery, and viability were 90, 19, and 85 percent, respectively. Gram stain, sterility, and endotoxin testing were all within acceptable limits for established lot release. Compared to the resting processed cells, IL-2 activation significantly increased the function of cells in cytotoxicity assays.CONCLUSION:Clinical-scale production of NK cells is efficient and can be performed under GMPs. The purified NK cell product results in high NK cell purity with minimal contamination by T cells, monocytes, and B cells, but it requires more time for processing and results in a lower NK cell recovery when compared to NK cell enrichment with CD3 cell depletion alone. Additional laboratory studies and results from clinical trials will identify the best source and type of NK cell product.

BACKGROUND:Errors and accidents, or deviations from standard operating procedures, other policy, or regulations must be documented and reviewed, with corrective actions taken to assure quality performance in a cellular therapy laboratory. Though expectations and guidance for deviation management exist, a description of the framework for the development of such a program is lacking in the literature. Here we describe our deviation management program, which uses a Microsoft Access database and Microsoft Excel to analyze deviations and notable events, facilitating quality assurance (QA) functions and ongoing process improvement.METHODS:Data is stored in a Microsoft Access database with an assignment to one of six deviation type categories. Deviation events are evaluated for potential impact on patient and product, and impact scores for each are determined using a 0- 4 grading scale. An immediate investigation occurs, and corrective actions are taken to prevent future similar events from taking place. Additionally, deviation data is collectively analyzed on a quarterly basis using Microsoft Excel, to identify recurring events or developing trends.RESULTS:Between January 1, 2001 and December 31, 2001 over 2500 products were processed at our laboratory. During this time period, 335 deviations and notable events occurred, affecting 385 products and/or patients. Deviations within the 'technical error' category were most common (37%). Thirteen percent of deviations had a patient and/or a product impact score > or = 2, a score indicating, at a minimum, potentially affected patient outcome or moderate effect upon product quality.DISCUSSION:Real-time analysis and quarterly review of deviations using our deviation management program allows for identification and correction of deviations. Monitoring of deviation trends allows for process improvement and overall successful functioning of the QA program in the cell therapy laboratory. Our deviation management program could serve as a model for other laboratories in need of such a program.